Regulation of Carcinogenesis by IL-5 and CCL11: A Potential Role for in Tumor Immune Surveillance

This information is current as Ljubov Simson, Julia I. Ellyard, Lindsay A. Dent, Klaus I. of October 1, 2021. Matthaei, Marc E. Rothenberg, Paul S. Foster, Mark J. Smyth and Christopher R. Parish J Immunol 2007; 178:4222-4229; ; doi: 10.4049/jimmunol.178.7.4222 http://www.jimmunol.org/content/178/7/4222 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Carcinogenesis by IL-5 and CCL11: A Potential Role for Eosinophils in Tumor Immune Surveillance1

Ljubov Simson,* Julia I. Ellyard,* Lindsay A. Dent,† Klaus I. Matthaei,‡ Marc E. Rothenberg,§ Paul S. Foster,‡ Mark J. Smyth,¶ and Christopher R. Parish2*

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor devel- opment and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 trans- genic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key that recruits eosinophils into tissues, and the -deficient mouse strains, IL-5/CCL11؊/؊ and ⌬dblGATA. It was found that Downloaded from MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11؊/؊ BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/ CCL11؊/؊ and ⌬dblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our http://www.jimmunol.org/ data support a potential role for the eosinophil as an effector cell in tumor immune surveillance. The Journal of Immunology, 2007, 178: 4222–4229.

he requirement for a cooperative interaction between the NK cells in this process (2). In addition, studies have highlighted innate and adaptive immune systems for effective tumor the role of CD4ϩ T cells in both the activation of innate effector T immunotherapy and tumor immune surveillance is widely cells in the primary response and subsequent cooperation with in- recognized. Most importantly, the host immune status has been nate cells in tumor cell killing (3). identified as critical in controlling the spread and growth of met- The role of Th2-mediated events in tumor immune surveillance astatic tumors (1). Until now, many tumor immune surveillance and, in particular, the eosinophilic granulocyte, has not been thor- by guest on October 1, 2021 studies have focused on the role of the adaptive dendritic cell- oughly investigated. Eosinophils have been implicated in resis- ϩ driven CD8 CTLs response in tumor elimination following rec- tance to tissue-invasive parasitic helminths and in the pathology of ognition of tumor Ags presented by MHC class I molecules on atopic conditions including asthma and atopic dermatitis; however, transformed cells. It should be noted, however, that the ability of the true immunological role(s) of this cell have yet to be eluci- the host to generate an effective innate immune response is fun- dated. Eosinophils are commonly found in infiltrates of many dif- damental to the generation of an adaptive CTL-response, and pre- ferent cancers (4), and controversy exists as to whether they are vious studies have confirmed the importance of macrophages and passive bystanders or active cellular agents in host immune re- sponses. Several in vivo studies have found a strong link between tumor eradication and eosinophil recruitment. Studies by Tepper *Division of Immunology and Genetics, John Curtin School of Medical Research, et al. (5) using IL-4-transfected tumor cell lines found that IL-4 Australian National University, Canberra, Australian Capital Territory, Australia; †School of Molecular and Biomedical Science, University of Adelaide, Adelaide, displayed potent antitumor activity, primarily mediated by eosin- South Australia, Australia; ‡Division of Molecular Bioscience, John Curtin School of ophils and cooperative killing by macrophages. The antitumor ef- Medical Research, Australian National University, Canberra, Australian Capital Ter- fect of transplanted IL-4-secreting tumor cells was associated with ritory, Australia; §Division of and Immunology, Cincinnati Children’s Hos- pital Medical Center, Cincinnati, OH 46229; and ¶Cancer Immunology Program, Sir the IL-4-induced local production of the eosinophil-specific che- Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East mokine, CCL11 (eotaxin-1) (6). Previous studies in our laboratory Melbourne, Victoria, Australia ϩ have demonstrated that tumor-specific CD4 T cells, exhibiting a Received for publication January 5, 2006. Accepted for publication January 8, 2007. profile characteristic of Th2 cells, were capable of clear- The costs of publication of this article were defrayed in part by the payment of page ing established lung and visceral metastases of a CTL-resistant charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. melanoma, with degranulating eosinophils within the tumors in- 1 This work was supported by Program Grants to C.R.P. (209618), M.J.S. (251608), ducing regression in a CCL11-dependent manner (7). P.S.F. and K.I.M. (224207), a Project Grant to L.A.D. (157964) from the National Expressed constitutively in many tissues (8), CCL11 is re- Health and Medical Research Council of Australia, and a Peter Doherty Biomedical Fellowship (National Health and Medical Research Council) to L.S. garded as one of the most potent and specific effector molecules targeting eosinophils, its pleiotropic effects including the bone 2 Address correspondence and reprint requests to Dr. Christopher R. Parish, John Curtin School of Medical Research, Division of Immunology and Genetics, Mills marrow maturation and release of eosinophils (9) and the di- Road Acton, Canberra, Australian Capital Territory, Australia. E-mail address: rected trafficking of eosinophils into tissues (10). During Th2- [email protected] mediated inflammatory conditions, epithelial cells and fibro- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 blasts provide the main source of CCL11. In contrast, IL-5 is www.jimmunol.org The Journal of Immunology 4223 produced by activated T lymphocytes, mast cells, and NK cells, Research and the Peter MacCallum Cancer Centre: CCL11Ϫ/Ϫ mice (17) and has several functions in eosinophil biology. Although initially backcrossed to a BALB/c or C57BL/6 background for 10 and 11 genera- identified as a B cell growth and differentiation factor (11), studies tions, respectively; BALB/c and C57BL/6 IL-5 Tg mice (13, 21) back- 3 Ϫ/Ϫ crossed to their respective backgrounds for 20 generations; BALB/c in vitro (12) and in IL-5 transgenic (Tg) (13) and IL-5 mice ⌬dblGATA mice backcrossed for 10 generations, followed by four inter- (14) have established that the major role for IL-5 is in eosinophi- crosses; and BALB/c IL-5/CCL11Ϫ/Ϫ mice backcrossed for 20 generations lopoiesis (12), in eosinophil trafficking and survival (15), and in as IL-5Ϫ/Ϫ followed by backcrossing with CCL11Ϫ/Ϫ mice for an addi- eosinophil activation, and the subsequent release of cytotoxic gran- tional 10 generations. Mice were treated according to the Australian Na- tional University and Peter MacCallum Cancer Centre Animal Welfare ule (16). In the present study, we were interested in in- guidelines and were housed in an approved containment facility. vestigating the role of Th2-mediated signals and eosinophils in tumor immune surveillance, using the methylcholanthrene (MCA)- Fibrosarcoma induction by MCA induced fibrosarcoma model. To address this question, we used BALB/c and B6 WT, CCL11Ϫ/Ϫ and IL-5 Tg, and BALB/c IL-5/ genetically modified mice known to express low and high endog- CCL11Ϫ/Ϫ and ⌬dblGATA mice were inoculated s.c. in the left hind leg enous levels of eosinophils, namely CCL11Ϫ/Ϫ (17) and IL-5 Tg with 0.1 ml of corn oil containing 1–400 ␮g of MCA (Sigma-Aldrich) and mice (13), and the eosinophil-deficient mouse strains, IL-5/ monitored weekly over a 26-wk period for the establishment and growth of fibrosarcomas. Mean tumor size was calculated as the product of the two Ϫ/Ϫ ⌬ CCL11 and dblGATA. perpendicular axes of the tumor (cm2), measured using Vernier calipers. Notably, IL-5 mRNA expression, regulated by the dominant Small protrusions (Ͻ2 mm in diameter), histologically determined to be controlling region of the human CD2 in IL-5 Tg mice, has encapsulated MCA, were evident in the majority of mice in all low-dose been determined to be restricted to s.c. lymph nodes, the thymus, (e.g., 5 ␮g) MCA groups. Therefore, protrusions Ͼ4 mm in diameter and demonstrating progressive growth over three or more weeks were recorded bone marrow, and Peyer’s patches, with no mRNA expression de-

as fibrosarcoma positive. Mice were sacrificed before fibrosarcomas reach- Downloaded from tected in the skeletal muscle, heart, or brain (13). The resulting ing 1.5 cm2 or, alternatively, at the conclusion of the 180-day experiment. phenotype of the IL-5 Tg mouse is characterized by 25–50% eo- Fibrosarcomas were aseptically removed and fixed in 10% phosphate-buff- sinophils in the circulating leukocyte pool (compared with Ͻ3% in ered formalin for 72 h before histological staining. wild type (WT)), and elevated levels of eosinophils in the spleen, Histological analysis bone marrow, and peritoneal cavity (13), thus providing an ideal model for studying the role of these cells in tumor immune sur- Fibrosarcomas were sectioned and stained with Carbol’s chromotrope-he-

matoxylin for identification of eosinophils. Eosinophils were quantified (10 http://www.jimmunol.org/ veillance. In stark contrast, the targeted disruption of the CCL11 fields/section, one section/animal at ϫ400 magnification) and represented gene results in the impaired recruitment of eosinophils, highlighted as the mean Ϯ SEM of five mice from each group unless otherwise spec- by a 2- to 3-fold decrease in circulating eosinophils in naive and ified. Photomicrographs of histological preparations were taken at ϫ100 to allergic mice when compared with WT cohorts (17). In addition ϫ400 magnification (as specified for each figure) using a BX50 micro- scope with CCD capability (Olympus). to the impaired eosinophil recruitment model, we used the eosi- nophil-deficient IL-5/CCL11Ϫ/Ϫ and ⌬dblGATA mouse strains. Lectin perfusion and vascular staining Deletion of the high-affinity GATA-binding site in the GATA-1 Fluorescein (FITC)-labeled lectin (Lycopersicon esculentum; Sigma- promoter has been found to lead to the selective loss of the eosi- Aldrich) perfusion was performed as described previously (22). Briefly, nophil-lineage (18) and a subsequent reduction in airways remod- mice were anesthetized, injected i.v. with 50 ␮g of FITC-labeled lectin, by guest on October 1, 2021 eling associated with chronic asthma (19). Mice deficient in both which was allowed to circulate for 10 min, and heart perfusion was per- IL-5 and CCL11 provide another useful model for eosinophil de- formed with 10% zinc-buffered formalin. Tumors were removed, fixed for 4 h in 4% paraformaldehyde, processed through 30% sucrose, and then letion, being eosinopoiesis-deficient and eosinophil recruitment embedded in Tissue-Tek OCT freezing medium (Sakura Finetek). Sections impaired, resulting in no blood or tissue eosinophilia in response to (35 ␮m) were cut and lectin staining was visualized using the Olympus allergic modeling (20). fluorescence microscope. In this study, a significant reduction in tumor establishment and Measurement of in vitro eosinophil-mediated tumor eradication growth was identified in IL-5 Tg mice that directly correlated with Preparation of eosinophils. OVA-activated eosinophils were generated in a high level of eosinophil recruitment to both the tumor and sur- BALB/c IL-5 Tg mice by i.p. sensitization with 50 ␮g of OVA/10% alu- rounding connective tissue. This result was substantiated by an minum hydroxide in normal saline on day 0, followed by i.p. challenge elevated tumor incidence and reduced influx of eosinophils into with 50 ␮g of OVA in normal saline on days 12, 13, and 14 before har- tumors observed in CCL11Ϫ/Ϫ mice of the BALB/c background. vesting eosinophils on day 15. Eosinophils were harvested from the peri- Furthermore, studies in eosinophil-deficient IL-5/CCL11Ϫ/Ϫ and toneal cavity by washing the cavity with HBSS and purified by sorting on ⌬ a BD FACSVantage (BD Biosciences) based on forward and side scatter dblGATA mice confirmed the correlation of high tumor incidence parameters and polarized light. The purity of the eosinophil-enriched pop- with an absence of eosinophils within tumors and the surrounding ulation was Ͼ95% as determined by differential staining with May- connective tissue. These novel observations suggest that the role of Gru¨wald Giemsa. Th2-derived and, in particular, of eosinophils warrants Coculture of eosinophils with MCA-induced fibrosarcoma cells. To further investigation to clarify their role in tumor immune measure eosinophil cytotoxic activity against MCA-induced fibrosarcoma cells in vitro, fibrosarcoma cells were seeded into a 96-well plate at 4.15 ϫ surveillance. 103 cells/well in F15/10% FCS and grown for 20–24 h before coculturing with sort-purified eosinophils in RPMI 1640/10% FCS alone or supple- Ϫ Materials and Methods mented with PMA (10 7 M) at various E:T ratios. Thirty hours after co- culture, 0.5 ␮Ci of tritiated ([3H])-thymidine was added to each culture Mice well in 20 ␮l of RPMI 1640/10% FCS and [3H]thymidine incorporation Six- to 9-wk-old BALB/c and C57BL/6 (B6) WT male mice were obtained measured for 18 h. At the end of the culture period, cell culture plates were from the specific pathogen-free facilities of either the John Curtin School centrifuged and cell supernatants discarded. Cells were washed once with of Medical Research (Australian National University, Canberra, Australia) PBS, then deadhered using 100 ␮l/well trypsin/EDTA at 37°C for 7 min. or the Walter and Eliza Hall Institute of Medical Research. The following Cell cultures were then stored at Ϫ20°C until harvesting. genetically modified mice, all 6- to 9-wk-old males, were bred and main- Measuring [ 3H]thymidine incorporation. Using a Filtermate 196 har- tained by the Gene Targeting Facility at the John Curtin School of Medical vester, cell cultures were harvested onto glass fiber filters (Packard Bio- science). Filters were dried and placed in Omnifilter plates, 20 ␮lofMi- croscint-O scintillation fluid was added to each well, and the plate was 3 3 Abbreviations used in this paper: Tg, transgenic; MCA, methylcholanthrene; WT, sealed with TopSeal-A adhesive film (PerkinElmer). [ H]Thymidine incor- wild type; HPF, high-powered field. poration was measured using a TopCount NXT (PerkinElmer). 4224 POTENTIAL ROLE FOR EOSINOPHILS IN TUMOR IMMUNE SURVEILLANCE

Statistical analysis Statistically significant differences were analyzed by using the Mann- Whitney U test for tumor growth rate data, with p Ͻ 0.05 considered as significant. A paired Student’s t test was used to calculate the statistical significance of the tumor incidence data. Significant differences between the eosinophil contents of tumors were calculated using unpaired Student’s t tests.

Results IL-5 overexpression protects mice from MCA-induced fibrosarcoma establishment and growth We were interested in the potential role for Th2-mediated events, in particular eosinophils, in tumor immune surveillance. The IL-5 Tg mouse was used in these studies due to its high constitutive level of circulating eosinophils, whereas CCL11Ϫ/Ϫ mice were used to determine the impact of impaired eosinophils recruitment. Initially, we examined the effect of MCA dose on tumor incidence in WT, IL-5 Tg, and CCL11Ϫ/Ϫ BALB/c mice (Fig. 1A). It was noted that, across the MCA dose range examined, IL-5 Tg mice were uniformly more resistant to MCA-fibrosarcoma induction Downloaded from compared with WT mice, with a 50% reduction in tumor incidence in the IL-5 Tg group at the 100 ␮g MCA dose. In contrast, CCL11Ϫ/Ϫ mice were consistently more sensitive to chemical car- cinogenesis than their WT controls. Indeed, at the 100 ␮g dose 90% of CCL11Ϫ/Ϫ mice, compared with only 60% of WT mice, had developed fibrosarcomas during the course of the 24 wk of the http://www.jimmunol.org/ experiment. Furthermore, at the lowest MCA dose tested (1 ␮g), only CCL11Ϫ/Ϫ mice developed tumors. We next examined larger cohorts of mice at a low dose of MCA (5 ␮g) to confirm statistical significance and to elucidate any genetic background-specific ef- fects by using mice with either the Th2-oriented BALB/c or Th1- oriented C57BL/6 backgrounds. It was confirmed that only 6% (1 of 16) of BALB/c IL-5 Tg mice and 20% (4 of 19) of C57BL/6 IL-5 Tg mice developed progressively growing fibrosarcomas by guest on October 1, 2021 compared with 40% (8 of 20) of BALB/c WT and 65% (13 of 20) of C57BL/6 WT controls during the 24-wk experiment (Fig. 1B). The difference in tumor incidence between IL-5 Tg and WT groups of both genetic backgrounds was found to be highly significant ( p Ͻ 0.001) from wk 16 in the BALB/c background and wk 18 in FIGURE 1. Overexpression of IL-5 protects mice from MCA-induced C57BL/6 mice (Fig. 1B). Furthermore, the onset of fibrosarcomas fibrosarcomas, whereas lack of CCL11 renders animals more susceptible to was considerably delayed in BALB/c IL-5 Tg mice, being wk 18 MCA-induced carcinogenesis. A, Dose-dependent increase in incidence of in BALB/c IL-5 Tg mice compared with wk 12 in the syngeneic MCA-induced fibrosarcomas is limited in IL-5 Tg BALB/c mice and in- Ϫ/Ϫ WT group. In stark contrast, the BALB/c CCL11 cohort dis- creased in CCL11Ϫ/Ϫ BALB/c mice relative to syngeneic WT animals. played a significant ( p ϭ 0.017) increase in tumor incidence (72%, Groups of WT, CCL11Ϫ/Ϫ, and IL-5 Tg BALB/c mice were injected s.c. in 13 of 18 mice) and an earlier onset of fibrosarcoma development the hind flank with 1–100 ␮g of MCA diluted in 0.1 ml of corn oil and (ie, wk 10). Interestingly, no significant change in tumor incidence observed weekly for 24 wk for the development of tumors. Tumor inci- or onset was observed in C57BL/6 CCL11Ϫ/Ϫ mice (Fig. 1B), dence at 24 wk was recorded as a percentage of tumor-positive mice in implying that the genetic background of mice influences the effect each group. Significant differences between genetically modified groups of CCL11 deficiency. and WT were determined at 24 wk by a Fisher’s exact test as shown. B, Incidence of fibrosarcoma development over time in IL-5 Tg and Further evaluation of tumor progression highlighted a strong Ϫ/Ϫ ␮ CCL11 mice with BALB/c and C57BL/6 backgrounds. Mice were in- reduction in the growth rate of MCA-induced fibrosarcomas (5 g jected s.c. in the hind flank with 5 ␮g of MCA diluted in 0.1 ml of corn oil dose) with IL-5 Tg mice of both genetic backgrounds (Fig. 2A). and observed weekly for tumor development over the course of 24 wk. Mean growth rates were determined by comparing the primary Significant differences between genetically modified groups and WT were fibrosarcoma growth phase of individual tumors in each group determined for the entire time course by a paired Student’s t test. Number and representative trend lines were produced. Using the Mann- of mice in each treatment group is shown in parentheses. Whitney U test, it was found that, irrespective of the genetic back- ground (ie, BALB/c or C57BL/6), the growth rate of MCA-in- duced tumors in the IL-5 Tg mice was significantly slower than in mice than in WT BALB/c mice ( p Ͻ 0.05). The tumor growth rate WT or CCL11Ϫ/Ϫ mice (Fig. 2B). In contrast, when comparing the curves (Fig. 2A) also highlight the slow progress of tumor devel- fibrosarcoma growth rates over the entire time course of the ex- opment within the IL-5 Tg mice of both backgrounds. In particu- periment, no significant differences were observed between the lar, the BALB/c IL-5 Tg group displayed several tumors (3 of 16) growth rates of the tumors induced in the CCL11Ϫ/Ϫ and WT exhibiting the arrested growth characteristic of tumor dormancy groups. However, once tumors reached 0.5 cm2 in size, their and in one mouse (1 of 16), tumor regression. To further highlight the growth rate was substantially (28%) faster in BALB/c CCL11Ϫ/Ϫ growth retardation of these tumors, ex vivo cultures of fibrosarcoma The Journal of Immunology 4225 Downloaded from

FIGURE 3. Eosinophil content of MCA-induced tumors from WT, CCL11Ϫ/Ϫ, and IL-5 Tg mice. Fibrosarcomas induced by 5 ␮gofMCA (Fig. 1B) in WT, CCL11Ϫ/Ϫ, and IL-5 Tg mice of BALB/c and C57BL/6 background were excised at 24 wk and examined histologically for eosin- ophil content. A, Representative fields from the fibrosarcomas were stained

using Carbol’s chromotrope-hematoxylin for the identification of eosinophils http://www.jimmunol.org/ (ϫ400 magnification). B, Eosinophil content of tumors from different exper- imental groups were quantified, and data are presented as mean eosinophil number per 10 high-powered fields (HPFs) Ϯ SEM of tumors from five mice of each WT and CCL11Ϫ/Ϫ group being examined. In the IL-5 Tg groups, only progressive tumors were examined, hence one mouse of BALB/c and four mice of C57BL/6 background were assessed. Significant differences between Ͻ ء FIGURE 2. Reduced growth rates of MCA-induced tumors in IL-5 Tg groups were calculated using an unpaired Student’s t test ( , p 0.001). mice. Groups of WT, CCL11Ϫ/Ϫ, and IL-5 Tg mice (number of mice in parentheses) of BALB/c and C57BL/6 background were injected s.c. in the Ϫ/Ϫ by guest on October 1, 2021 hind flank with 5 ␮g of MCA diluted in 0.1 ml of corn oil and observed C57BL/6 CCL11 mice when compared with WT syngeneic weekly over 24 wk for tumor growth. A, Tumors Ͼ2 mm in diameter were mice (Fig. 3B). Ϫ/Ϫ measured weekly and the size was calculated as the product of two per- The presence of residual eosinophils in the tumors of CCL11 pendicular diameters (cm2). Each line with ࡗ represents data for an indi- cohorts of both genetic backgrounds (Fig. 3, A and B) suggests that vidual animal, whereas the lines of best fit for each group (demonstrating other /cytokines are also involved in the recruitment of mean growth rate) are represented by a solid trend-line. B, Statistical analysis eosinophils in the tumor environment. However, the relative re- of differences between trend lines was tested with the Mann-Whitney U test. duction in eosinophil numbers observed in the tumors of the BALB/c CCL11Ϫ/Ϫ group (Fig. 3B), when compared directly to the tumors of WT BALB/c mice, correlates with the increase in lines from IL-5 Tg mice were extremely difficult to propagate com- tumor incidence and more rapid growth rate of established tumors pared with the rapid growth of fibrosarcoma lines derived from the observed in this group (Fig. 1B). In contrast, the maintenance of tumors of WT and CCL11Ϫ/Ϫ mice (data not shown). eosinophil numbers equivalent to WT levels in the C57BL/6 CCL11Ϫ/Ϫ tumors directly correlates with the lack of a significant Tumor-associated eosinophilia correlates with IL-5-mediated change in tumor incidence and growth rate in these mice (Fig. 1B). tumor suppression Collectively, these data suggest that differences in the tumor inci- Ϫ Ϫ Consistent with observations made in many human tumors (4) and dence in CCL11 / BALB/c and C57BL/6 mice is due to the murine tumor studies, histological examination revealed the pres- relative importance of CCL11 in recruiting eosinophils into tissues ence of a small population of eosinophils in the tumors of WT in these two mouse strains rather than the Th2-oriented nature of mice of both the BALB/c and C57BL/6 backgrounds. When com- the BALB/c background vs the Th1-oriented nature of the pared with syngeneic WT mice, tumor-infiltrating eosinophil num- C57BL/6 background. bers were increased 20-fold in the BALB/c IL-5 Tg progressive tumor and up to 9-fold in the C57BL/6 IL-5 Tg tumors (Fig. 3B; Persistent tumor encapsulation and ongoing immune p Ͻ 0.001 compared with WT), revealing a strong correlation surveillance in IL-5 Tg mice ,ء with reduced tumor incidence (Fig. 1), delayed tumor establish- Low-dose (5 ␮g) MCA BALB/c mice of the WT, CCL11Ϫ/Ϫ, and ment (Fig. 2), and slower growth rate of tumors in these mice (Fig. IL-5 Tg backgrounds displayed varying levels of MCA encapsu- 2). Conversely, although not quite statistically significant, eo- lation in the first 4 wk after MCA injection (data not shown). The sinophil numbers were reduced by approximately one-third in majority of mice in the WT and CCL11Ϫ/Ϫ groups that had ini- BALB/c CCL11Ϫ/Ϫ tumors (Fig. 3B), whereas, consistent with tially displayed partial MCA encapsulation proceeded to develop no significant change in tumor incidence (Fig. 1B), minimal rapidly outgrowing fibrosarcomas, with tumor cells penetrating the change was detected in eosinophil numbers in the tumors of surrounding connective tissue layers to the overlying epidermis 4226 POTENTIAL ROLE FOR EOSINOPHILS IN TUMOR IMMUNE SURVEILLANCE Downloaded from http://www.jimmunol.org/

FIGURE 4. Tumor encapsulation and poor tumor outgrowth is evident in IL-5 Tg BALB/c mice following low-dose (5 ␮g) MCA inoculation. Mice were injected s.c. in the hind flank with 5 ␮g of MCA diluted in 0.1 FIGURE 5. Eosinophil depletion in IL-5/CCL11Ϫ/Ϫ and ⌬dblGATA ml of corn oil, and MCA-induced fibrosarcomas were monitored over the mice is associated with increased susceptibility to MCA-induced carcino- 24 wk of the experiment. Upon completion of the experiment (wk 24), genesis. A higher incidence of MCA-induced fibrosarcomas is observed in poorly growing fibrosarcomas from the IL-5 Tg BALB/c group were his- IL-5/CCL11Ϫ/Ϫ and ⌬dblGATA BALB/c mice relative to syngeneic WT tologically examined and compared with WT and “progressive” IL-5 Tg by guest on October 1, 2021 animals at both low and high doses of MCA. Groups of WT, IL-5/ tumors following staining with Carbol’s chromotrope-hematoxylin. A, CCL11Ϫ/Ϫ, and ⌬dblGATA BALB/c mice were injected s.c. in the hind Representative tumor in BALB/c WT mice viewed at ϫ100 (i) and sur- flank with 5 or 100 ␮g of MCA diluted in 0.1 ml of corn oil and observed rounding connective tissue at ϫ400 (ii) magnification (F, fibroblasts and weekly for 24 wk for the development of tumors. Tumor incidence over the connective tissue; SM, smooth muscle; D, dermal tissue; E, epidermal tis- 24 wk was recorded as the percentage of tumor-positive mice in each sue; T, tumor tissue). B, Encapsulated “nonprogressive” fibrosarcoma rep- group. Significant differences between groups were determined for the en- resentative of 4 of 16 mice in the BALB/c IL-5 Tg group (5 Ϯ 1mm tire time course using a paired Student’s t test as shown. diameter) viewed at ϫ40 (i) and surrounding fibrotic capsule at ϫ400 (ii) magnification (FC, fibrotic capsule; F, fibroblast and connective tissue; M, MCA injection site; SM, smooth muscle; T, tumor). Carbol’s chromotrope- pying ϳ70% of the encapsulated area, surrounding a small cluster stained eosinophils appear with distinctive red cytoplasmic granules in (ii). of tumor cells. Tumor growth had been arrested, and in one case C, Eosinophil influx was compared between BALB/c WT, progressive IL-5 Tg, and nonprogressive IL-5 Tg tumors from the 5 ␮g of MCA groups. regression induced, from the time of capsule formation at 6–12 wk Eosinophils were quantified (10 fields/tumor/four mice per group at ϫ400 throughout the course of the experiment (Fig. 3). As previously magnification) within the tumor and surrounding connective tissue and mentioned, we were unable to successfully culture cell lines from represented as mean per 10 HPFs Ϯ SEM of four mice. Significant dif- these dormant IL-5 Tg mouse “tumors,” suggesting poor cellular ferences between groups were calculated using an unpaired Student’s t test growth in vivo and ex vivo. The most striking feature of these p Ͻ 0.001). lesions was a persistently high level of eosinophils within both the ,ء) tumor stroma and surrounding connective tissue (Fig. 4B). Eosin- ophil numbers in the connective tissue (fibrotic capsule) surround- (Fig. 4Ai) and underlying skeletal muscle (data not shown). His- ing the nonprogressive tumor were found to be 100-fold higher tological analysis revealed a low level of infiltrating eosinophils than observed in WT syngeneic mice and 3.5-fold higher than the and other leukocytes (Fig. 4Aii). This result suggests that any pro- progressive IL-5 Tg BALB/c tumor. Visualization of tumor vas- tective effect mediated by the surrounding connective and fibrous culature using FITC-lectin perfusion showed no significant differ- tissue and associated recruited innate inflammatory cells was lim- ences between WT and nonprogressive tumors in the IL-5 Tg ited by the host’s inability to eradicate cells transformed progres- strain (data not shown). sively by the carcinogen. In contrast, in the IL-5 Tg groups encapsulation persisted Eosinophil deficiency correlates with an increased tumor throughout the 24 wk of the experiment. Upon histological anal- incidence ysis (Fig. 4Bi) it was revealed that in 4 of 16 BALB/c IL-5 Tg To further investigate the potential role of the eosinophil in tumor mice, the persistent fibrotic capsule (size 5 Ϯ 1 mm) contained a immune surveillance, the eosinophil-deficient IL-5/CCL11Ϫ/Ϫ and high influx of eosinophils and other leukocytes (Fig. 4Bii) occu- ⌬dblGATA BALB/c mutant strains were treated with MCA to The Journal of Immunology 4227 Downloaded from FIGURE 6. Eosinophil content of MCA-induced tumors from WT, IL- 5/CCL11Ϫ/Ϫ, and ⌬dblGATA mice. Fibrosarcomas induced by 5 ␮gof MCA (Fig. 5) in WT, IL-5/CCL11Ϫ/Ϫ, and ⌬dblGATA mice of BALB/c background were excised at 24 wk and examined for eosinophil content. A, Representative fields from the fibrosarcomas were stained using Carbol’s chromotrope-hematoxylin for the identification of eosinophils (ϫ400 mag- nification). Representative eosinophils are indicated by arrows. B, Eosin- http://www.jimmunol.org/ ophil content of tumors from different experimental groups were quanti- fied, and data are presented as mean eosinophil number per 10 HPFs Ϯ SEM of tumors from five mice of each group examined.

induce fibrosarcomas. At the 5 and 100 ␮g dose of MCA, both strains showed a significant increase ( p Ͻ 0.005) in tumor inci- dence when compared with syngeneic WT controls (Fig. 5). In- by guest on October 1, 2021 deed, at the 5 ␮g dose, 58% of the IL-5/CCL11Ϫ/Ϫ and 82% of the ⌬dblGATA mice had developed fibrosarcomas during the 24-wk experiment, compared with an incidence of only 22% in WT mice. FIGURE 7. In vitro eosinophil-mediated killing of MCA-induced fibro- In addition, 90 and 94% of the IL-5/CCL11Ϫ/Ϫ and ⌬dblGATA sarcoma cells. A MCA-fibrosarcoma cell line, derived from a tumor es- ␮ mice, respectively, had developed tumors at the 100 ␮g MCA dose tablished in a WT BALB/c mouse following 5 g of MCA administration, was used to investigate the ability of eosinophils to mediate fibrosarcoma compared with only 61% of the WT cohort. Although there was no cell death. A, Bright field images (ϫ400 magnification) illustrate fibrosar- significant difference in tumor incidence between the IL-5/ coma viability following various treatments: i, fibrosarcoma alone; ii,fi- Ϫ/Ϫ ⌬ ␮ CCL11 and dblGATA cohorts at either 5 or 100 g MCA, the brosarcoma with unstimulated OVA-activated eosinophils at an E:T ratio ⌬dblGATA mice were particularly sensitive to MCA treatment, of 25:1; iii, fibrosarcoma with PMA (10Ϫ7 M)-stimulated OVA-activated with tumor incidence being extremely high at both the low and eosinophils at an E:T ratio of 25:1; iv, fibrosarcoma with PMA (10Ϫ7 high MCA doses, at 82 to 90%, respectively. M)-stimulated OVA-activated eosinophils at an E:T ratio of 50:1. B, Fi- A histological examination was performed on the connective brosarcoma cell viability was quantified using [3H]thymidine incorporation tissue surrounding the tumors, the tumor tissue, and necrotic cores following fibrosarcoma treatment with unstimulated or PMA-stimulated (data not shown), confirming a low infiltration of eosinophils OVA-activated eosinophils at E:T ratios of 12.5:1, 25:1, 50:1, and 100:1. throughout these regions in all WT mice (Fig. 6). However, no eosinophils were observed in these regions at either the 5 or 100 ␮g MCA dose in the IL-5/CCL11Ϫ/Ϫ and ⌬dblGATA cohorts (data not shown), revealing a strong correlation with reduced tumor in- MCA-induced tumor, following the administration of 5 ␮gof cidence in these groups (Fig. 5). As seen in the first series of MCA in a WT BALB/c mouse, was used as the target in these experiments (Fig. 1), WT mice exhibited MCA encapsulation in experiments, whereas OVA-stimulated eosinophils mimicking a the first 4 wk after MCA injection (data not shown), whereas sev- Th2-primed tumor environment were used as effector cells. PMA- eral animals in the IL-5/CCL11Ϫ/Ϫ and ⌬dblGATA groups dis- stimulated OVA-activated eosinophils were able to instigate an played MCA-encapsulation, characterized by a less severe protru- almost complete eradication of MCA-fibrosarcoma cells at target sion and thinner fibrotic capsule (data not shown), which ratios of 25:1 and above (Fig. 7, Aiii and Aiv and B). In addition, progressed to develop into rapidly outgrowing fibrosarcomas. unstimulated OVA-activated eosinophils also illustrated effective fibrosarcoma killing (Fig. 7Aii), with only 60-40% of fibrosarcoma Eosinophils can directly mediate fibrosarcoma killing in vitro cells remaining viable following coincubation with eosinophils at In conjunction with in vivo immune surveillance experiments, the E:T ratios of 50:1 and 100:1, respectively (Fig. 7B). These results ability of eosinophils to mediate direct killing of fibrosarcoma cells confirm that eosinophils can mediate the direct killing of MCA- in vitro was investigated. A fibrosarcoma cell line derived from a induced fibrosarcoma cells. 4228 POTENTIAL ROLE FOR EOSINOPHILS IN TUMOR IMMUNE SURVEILLANCE

Discussion parasitic model, Ab is not the main mechanism of recognition and The data reported in this study provides in vivo and in vitro evi- attachment of eosinophils to helminth larvae (L. A. Dent, unpub- dence that eosinophils play an important role in limiting carcino- lished data). genesis and/or the growth of chemically induced tumors. We hy- In vivo experiments using either an eotaxin-expressing mela- pothesize that the initial tissue damage incurred by the MCA noma (L. Simson and C. R. Parish, unpublished data) or an injection in BALB/c WT, CCL11Ϫ/Ϫ, IL-5 Tg, IL-5/CCL11Ϫ/Ϫ, eotaxin-expressing hepatocellular carcinoma (32) have revealed and ⌬dblGATA mice alike, induces a wound-healing inflammatory that although eosinophils are successfully recruited into the tu- response, inducing the deposition of extracellular matrix to form mors, there is little affect on tumor growth unless the tumors are the fibrotic capsule and supporting a strong influx of innate effector grown in IL-5 Tg recipients, confirming the potential importance cells (23). In WT, CCL11Ϫ/Ϫ, IL-5/CCL11Ϫ/Ϫ, and ⌬dblGATA of IL-5 in tumor eradication. In addition, our preliminary in vitro mice, this initial response is not sufficient to contain malignant results with eosinophils confirm the obligatory role of IL-5 and transformation, nor for the induction of an appropriate immune suggest other myeloid cells may be involved in tumor eradication surveillance response, and mutating cells rapidly overwhelm the (J. I. Ellyard, L. Simson, and C. R. Parish, unpublished data). It is host. However, in the IL-5 Tg mice, this initial inflammatory likely that eosinophils act in concert with other immune cells in the response favors the establishment and maintenance of an ap- immune surveillance process. For example, it is known that NKT propriate eosinophil-mediated antitumor response. The fibrotic cells suppress MCA-induced fibrosarcoma formation (2), and re- capsule surrounding the carcinogen, containing the extracellular cently NKT cells, in association with eosinophils, have been shown to contribute to allergen-induced airway inflammation in an matrix and associated glycosaminoglycans, provides the scaf- IL-4- and eotaxin-dependent manner (33, 34). fold for the formation of a strong IL-5 chemotactic gradient and Downloaded from In conclusion, our results expand considerably on previous find- maintenance of eosinophil-mediated tumor eradication (24). ings showing the ability of Th2-mediated immunity and eosino- Hence, eosinophils may also play a role in enhancing the MCA- phils to induce tumor suppression. Clarifying the role of eosino- encapsulation process, restricting the contact of surrounding phils in tumor immune surveillance has the potential for subepithelial tissue with MCA and resulting in a subsequent elucidating the general immune function of these cells as well as reduction in cellular mutations, as well as playing an active role establishing their role in tumor development and cancer

in the ongoing immune surveillance process. http://www.jimmunol.org/ immunotherapy. Hence, the relationship between eosinophils and other immune cells known to be involved in tumor immune surveillance is of considerable interest. In addition, the signals required for the re- Acknowledgments cruitment of eosinophils into the tumor environment and the sub- We thank the animal staff at the John Curtin School of Medical Research (J.C.S.M.R.) and Peter MacCallum Cancer Centre (P.M.C.C.), in particular sequent mechanisms used by eosinophils to induce tumor cell Wayne Damcevski and Shannon Griffiths, for the breeding of genetically death warrant further investigation. Although eosinophil recruit- modified mice. We would also like to thank Jeremy Swann from the ment into the tumor may be partially dependent on the basal ex- P.M.C.C. for expert technical assistance and Anne Prins from the J.C. pression of eosinophil-specific chemokines, including CCL11 (8), S.M.R. Microscopy Unit for assistance with histological preparations. from the fibrosarcoma itself, both the innate and adaptive immune by guest on October 1, 2021 responses are likely to be involved in eosinophil recruitment and Disclosures activation. The potential cooperation between eosinophils and The authors have no financial conflict of interest. other innate cells, namely NK cells, NKT cells, and macrophages in eosinophil recruitment, is of particular interest. The role of NK References cells in tumor immune surveillance, including MCA-induced fi- 1. Dunn, G. P., A. T. Bruce, H. Ikeda, L. J. Old, and R. D. Schreiber. 2002. Cancer brosarcomas, is well characterized (2, 25). NK cells mediate spon- immunoediting: from immunosurveillance to tumor escape. Nat. Immunol. 3: 991–998. taneous cytotoxicity against MHC class I-deficient tumor cells and 2. Smyth, M. J., K. Y. Thia, S. E. Street, E. Cretney, J. A. Trapani, M. 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